Replicate arrays of biological agents have been used to facilitate parallel testing of many samples. For example, sterile velvet cloths and a piston-ring apparatus has long been used to make replicates of bacterial and yeast colonies to agar plates each containing a different growth medium, as a means of rapidly screening a large number of independent colonies for different growth phenotypes (Lederberg and Lederberg, J. Bacteriol. 63 :399, 1952). Likewise, 96-well microtiter plates are used to organize and store in an easily accessed fashion large numbers of e.g. cell lines, virus isolates representing recombinant DNA libraries, or monoclonal antibody cell lines.
The advent of large scale genomic projects and the increasing use of molecular diagnostics has necessitated the development of large volume throughput methods for screening nucleic acids. Recently, methods have been developed to synthesize large arrays of short oligodeoxynucleotides (ODNs) bound to a glass or silicon surface that represent all, or a subset of all, possible nucleotide sequences (Maskos and Southern, Nucl. Acids Res. 20: 1675, 1992). These ODN arrays have been made used to perform DNA sequence analysis by hybridization (Southern et al., Genomics 13: 1008, 1992; Drmanac et al., Science 260: 1649, 1993), determine expression profiles, screen for mutations and the like. In all these uses, the ODNs are covalently attached to the surface of the substrate. However, some useful screening techniques and assays are not readily adaptable to a format in which ODNs are immobilized.
In particular, amplification of nucleic acids, notably the polymerase chain reaction (PCR) and its many variations, has found wide application to many different many biological problems and is not easily moved to a format where the ODNs are immobilized. In its standard format, PCR has two major limitations to its commercial utilization: the cost of reagents and the ability to automate the process. Reagent costs, especially DNA polymerase, can be lowered if the total volume of each reaction is decreased. An accurate and reliable means to array small volumes of reagents using a robotically controlled pin tool would miniaturize the reactions. Additional hurdles to moving amplification to an array format include preventing evaporation during heating and cooling cycles and preventing spreading and merging of the reactions on the array.
The present invention discloses methods and compositions for performing amplification and other enzymatic reactions in an array format without the need to immobilize the components, and further provides other related advantages.